A G→T splice site mutation of CRYBA1/A3 associated with autosomal dominant suture cataracts in a Chinese family.

PURPOSE
To identify the genetic defect in a five-generation Chinese family with congenital Y-suture cataracts.


METHODS
A five-generation Chinese family with inherited Y-suture cataract phenotype was recruited. Detailed family history and clinical data of the family were recorded. Candidate genes sequencing was performed to screen out the disease-causing mutation.


RESULTS
The congenital cataract phenotype of the family was identified as Y-suture cataract type by using slit-lamp photography. Direct sequencing revealed a G→T splice site mutation in crystallin, beta A1 (CRYBA1/A3).This mutation co-segregated with all affected individuals in the family and was not found in unaffected family members or 100 unrelated controls.


CONCLUSIONS
Our study identified a novel type of a splice site mutation in CRYBA1/A3 .The mutation was responsible for the congenital Y-suture cataracts in the family. This is the first report relating a G→T mutation of CRYBA1/A3 to congenital Y-suture cataract.

We applied a functional candidate approach testing the known cataract-causing genes in a Chinese family. A G→T splice mutation in CRYBA1/A3 was identified to be responsible for cataracts in the family. This is the first report to relate this mutation site to Y-suture cataracts also involving opacities of the nucleus.

METHODS
Family data: A five-generation Chinese family from Shandong Province with a history of cataracts was recruited from Beijing Tongren Hospital, Capital Medical University, Beijing, China. The research was approved by the ethics committee of Capital Medical University. Informed consent was obtained from all participants of the family. The study protocol followed the principles of the Declaration of Helsinki.
Detailed family medical history was recorded by interviewing the family members. All participating members underwent ophthalmic examination, including visual acuity, slit-lamp examination, intraocular pressure measurement, ultrasonography, and fundus examination of the dilated pupil. Slit-lamp photography was performed to document the phenotype of the cataracts in the patients. One hundred unrelated subjects without cataracts were recruited from the Ophthalmology Clinic of Beijing Tongren Hospital as normal controls and were given complete ophthalmologic examinations. None of the controls exhibited eye diseases except mild myopia. Genomic DNA preparation: About 2 ml of peripheral blood was collected from the family members who took part in the study. Genomic DNA was extracted from blood using the QIAamp Blood kit (Qiagen, Valencia, CA).
, and BFSP2 (GenBank NM_003571). Each exon and intron-exon junction of the genes were amplified by polymerase chain reaction (PCR) using previously published primer sequences (Table 1) [6]. Each reaction mix (25 μl) contained 20 ng of genomic DNA, 1× PCR buffer,1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 μM each of forward and reverse primers and 2.5 U of Taq DNA polymerase (Qiagen). A PCR program was performed for DNA amplifying: 95 °C for 5 min; followed by 35 cycles at 95 °C for 30 s, 57 °C-63 °C for 30 s (annealing temperature depending on different primer); 72 °C for 30 s; and a final extension at 72 °C for 10 min. The PCR products of the proband and one unaffected member were sequenced using an ABI3730 Automated Sequencer (PE Biosystems, Foster City, CA). The sequencing results were analyzed using Chromas 2.33 and compared with the reference sequence in the NCBI database. Then we screened the mutation in CRYBA1/A3 from the sample of the family members and 100 ethnically matched controls to confirm the mutation.

RESULTS
Clinical evaluation: Thirteen family members of a fivegeneration Chinese family with a history of cataracts participated in the study (six affected and seven unaffected individuals; Figure 1). All patients in this family had bilateral cataracts. Most patients experienced decreased visual acuity at 3-4 years old, and then their visual acuity decreased gradually until surgery was required. The proband, who was a 3-year-old girl, experienced a decrease in vision at 1.5 years old and had been diagnosed with bilateral cataracts at age 3. Slit-lamp examination revealed opacification of Y-sutue cataracts with opacities involving nucleus. The girl's best corrected visual acuity was 0.3/0.3. Her clinical features were similar to those of her uncle (IV:6) with peripheral cortical opacity ( Figure 2). His best corrected visual acuity was 0.3 / 0.4. The affected member IV:3, who was the father of the proband, had undergone cataract removal at age 8. Mutation analysis: Through direct gene sequencing of the coding regions of the candidate genes, we identified an IVS3+1 G→T substitution in the donor splice site of intron 3 in CRYBA1/A3 in all affected individuals ( Figure 3). However, we did not find this mutation in any unaffected family members or in the 100 unrelated controls. We did not find any other mutations in this family except for a few nonpathogenic single nucleotide polymorphisms (SNPs).  . In panel A, the mutation G→T was evident at the first base of intron 3, which was identified in all patients of the family, but was not found in the unaffected family members nor in the 100 unrelated control subjects.

DISCUSSION
In this study we identified a splice site mutation of CRYBA1/ A3 in a five-generation Chinese family with Y-suture opacities of the lens involving embryonic and fetal nuclei.
Sutural cataracts affect the sutural regions of the nucleus, at which the ends of the lens fiber cells meet. Sutural cataracts may occur in isolation or be associated with opacities involving other lens regions. There is some correlation between the pattern of expression of the mutant gene and the morphology of the resulting cataract.
So far, in the CRYBA1/A3 gene, three types of mutations have been associated with autosomal dominant cataracts. Our report of IVS3+1 G→T will be the fourth type of CRYBA1/ A3 mutation. The first one is the IVS3+1 G→A mutation. Regarding IVS3+1 G→A, in 1998 Kannabiran et al. [21] reported an Indian family with zonular cataracts with sutural opacities. In 2008, Devi et al. [22] reported another two Indian families with zonular lamellar cataracts. In 2004, Burdon et al. [15] reported an Australian family with Y-sutural cataracts. In 2010, Gu et al. [23] identified a Chinese family with posterior polar cataracts, which was the first time this mutation was found in the Chinese population. Also in 2010, Zhu et al. [18]reported a Chinese family with progressive childhood cataracts characterized by opacities in the fetal nucleus and peripheral cortex. The second type of mutation is IVS3+1 G→C. In 2000, Bateman et al. [16] reported a Brazilian family with varied clinical characteristics among the affected members. The affected individuals who were examined had pulverulent opacities in the embryonal nucleus and sutures and star-shaped, shieldlike, or radial opacities in the posterior embryonal nucleus. The third type of mutation is a 3-bp deletion at positions 276-281 in exon 4, which causes an in-frame deletion of a glycine residue at position 91 (ΔG91). In 2004, Qi et al. [24] identified a Chinese family with nuclear cataracts. In 2007, Lu et al. [25] reported two Chinese families with pulverulent congenital cataracts (Table  3).  CRYBA1/A3 consists of six exons encoding two proteins (βA3-crystallin and βA1-crystallin) by using an alternative translation initiation site. βA1/A3-crystallin consists of seven protein regions: four homologous (Greek key) motifs, a connecting peptide, and NH2-and COOH-terminal extensions.
In the CRYBA1/A3 gene, the first two exons encode the sequence of the N-terminal arm, and exons 3-6 encode the Greek key motifs 1-4 [27]. The G at position +1 of the 5′ (donor) splice site is highly conserved, and mutation of this base can be expected to disrupt the splice site [28]. In this study the mutation at IVS3+1 G→T can be expected to skip the donor splice junction, which may cause the wrong junction of the exons in CRYBA1/A3. This may result in premature termination of the polypeptide. In this condition, it would cause structural instability and disrupt the folding of the protein [21].
In conclusion, we have identified a new type IVS3+1 G→T mutation of the CRYBA1/A3 gene associated with Ysutural congenital cataracts in a Chinese family. This mutation supports the role of the CRYBA1/A3 gene in human cataract formation and provides more evidence of genetic heterogeneity of congenital cataracts.